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Abstract:

Provided is a chip or the like having a particulate concentrating
mechanism such as a mechanism that can selectively concentrate nucleated
red blood cells contained in maternal blood and derived from a fetus and
can collect the concentrated liquid rich in nucleated red blood cells,
and also provided is a nucleated red blood cell concentrating/collecting
method. A micro-channel chip for concentrating nucleated red blood cells
has an inlet-side channel, an outlet-side channel, and a separation
narrow channel provided between the inlet-side channel and the
outlet-side channel. The separation narrow channel has an inner wall
having a dimension through which non-nucleated red blood cells easily
pass and nucleated red blood cells hardly pass, and has a means for
deforming or moving part of the inner wall of the channel to have a
dimension through which nucleated red blood cells easily pass. The
following are also provided: a method for collecting a liquid in which
nucleated red blood cells obtained by using this chip are concentrated;
and a micro-channel chip for concentrating particulates other than for
concentrating nucleated red blood cells.

Claims:

1. A micro channel chip, employed to concentrate a granular material B
from a mixture of at least one type of granular material having arbitrary
grain diameter and arbitrary deformation property (referred to
hereinafter as granular material A) and at least one type of granular
material having a larger grain diameter than granular material A and less
deformability than granular material A (referred to as granular material
B hereinafter), comprising an inlet side channel, an outlet side channel,
and a separation-use narrow channel between the inlet side channel and
outlet side channel; wherein the separation-use narrow channel has an
inner wall, at least a part of which is comprised of a flexible film and
an air chamber is provided on the opposite side of the flexible film from
the air channel and the inner wall has dimensions permitting the ready
passage of granular material A and tending not to pass granular material
B; and comprising a means of deforming the flexible film by adjusting the
pressure in the air chamber to achieve dimensions facilitating the
passage of granular material B.

2. A micro channel chip for concentrating nucleated red blood cells,
comprising an inlet side channel, an outlet side channel, and a
separation-use narrow channel between the inlet side channel and outlet
side channel; wherein the separation-use narrow channel has an inner
wall, at least a part of which is comprised of a flexible film and an air
chamber is provided on the opposite side of the flexible film from the
channel and the inner wall has dimensions permitting the ready passage of
nonnucleated red blood cells and tending not to pass nucleated red blood
cells; and comprising a means of deforming or displacing a part of the
inner wall the flexible film by adjusting the pressure in the air chamber
to achieve dimensions facilitating the passage of nucleated red blood
cells.

3. The micro channel chip according to claim 2, wherein the inner wall of
the separation-use narrow channel has a vertical sectional height in the
channel in a range of 1 μm to 2 μm, a width in a range of 10 μm
to 10 cm, and a channel length in a range of 20 μm to 300 μm.

4. A micro channel chip for concentrating nucleated red blood cells,
comprising an inlet side channel, an outlet side channel, and a
separation-use narrow channel between the inlet side channel and outlet
side channel; wherein the separation-use narrow channel has an inner wall
of dimensions permitting the ready passage of nonnucleated red blood
cells and tending not to pass nucleated red blood cells; and wherein the
dimensions are such that the vertical sectional height in the channel is
in a range of 1 μm to 2 μm, the width is in a range of 10 μm to
10 cm, and the length of the channel is in a range of 20 μm to 300
μm.

5. The micro channel chip according to claim 1, wherein a plurality of
separation-use narrow channels are separated by spacers, the surface of
the spacer facing the channel on the outlet side is a curved surface that
is convex in shape on the outlet side channel side, and/or the surface of
the spacer facing the channel on the inlet side is a curved surface that
is convex in shape on the inlet side channel side.

6. The micro channel chip according to claim 1, wherein the inlet side
channel, outlet side channel, and separation-use narrow channel are built
into the chip, an inlet connecting to the inlet side channel is present
on the chip surface, an outlet connecting to the outlet side channel is
present, and an opening connecting to an air chamber is present.

7. The micro channel chip according to claim 1, wherein the inner wall of
each channel is surface treated with a coating to prevent cell adhesion
or with a coating to prevent nonspecific adhesion.

8. The micro channel chip according to claim 4, wherein the inner wall of
each channel is surface treated with a coating to prevent cell adhesion
or with a coating to prevent nonspecific adhesion.

9. A method for recovering a liquid in which nucleated red blood cells
have been concentrated, comprising: feeding a sample containing
nonnucleated red blood cells and nucleated red blood cells from the inlet
side channel of the micro channel chip according to claim 2; recovering
the liquid that has passed through the separation-use narrow channel from
the outlet side channel; causing a part of the inner wall of the channel
to deform or displace to assume dimensions allowing ready passage of the
nucleated red blood cells, and while in this state, feeding a recovering
liquid from the inlet side channel to recover a liquid rich in nucleated
red blood cells from the outlet side channel.

10. A method for recovering a liquid in which nucleated red blood cells
have been concentrated, comprising feeding a sample containing
nonnucleated red blood cells and nucleated red blood cells to the inlet
side channel of the micro channel chip according to claim 4; recovering
the liquid that has passed through the separation-use narrow channel from
the outlet side channel; feeding a recovering liquid from the inlet side
channel or outlet side channel, and recovering a liquid rich in nucleated
red blood cells from the outlet side channel or inlet side channel.

11. The method according to claim 9, wherein the sample containing the
nonnucleated red blood cells and nucleated red blood cells is a fraction
that has been recovered with a density of 1.070 g/mL to 1.095 g/mL by
density gradient centrifugation separation using Percoll.

12. The method according to claim 11, wherein the sample containing the
nonnucleated red blood cells and nucleated red blood cells comprises a
recovered fraction diluted with a saline solution having a sodium
chloride concentration consistent with physiological conditions.

13. The method according to claim 9, wherein in the course of feeding the
sample containing nonnucleated red blood cells and nucleated red blood
cells, the pressure-adjustable chamber is subjected to a positive
pressure relative to the separation-use narrow channel to prevent a part
of the inner wall of the channel from deforming in a concave manner to
the air chamber side.

14. The method according to claim 9, wherein the pressure in the air
chamber is reduced relative to the separation-use narrow channel to cause
a part of the inner wall of the channel to deform in a concave manner to
the air chamber side to achieve dimensions permitting the ready passage
of nucleated red blood cells.

15. The method according to claim 9, wherein the pressure in the air
chamber is reduced relative to the separation-use narrow channel to cause
a part of the inner wall of the channel to deform in a concave manner to
the air chamber side to achieve dimensions permitting the ready passage
of the liquid through the separation-use channel when the liquid is
introduced, during cleaning, or when removing air bubbles.

16. The micro channel chip according to claim 2, wherein a plurality of
separation-use narrow channels are separated by spacers, the surface of
the spacer facing the channel on the outlet side is a curved surface that
is convex in shape on the outlet side channel side, and/or the surface of
the spacer facing the channel on the inlet side is a curved surface that
is convex in shape on the inlet side channel side.

17. The micro channel chip according to claim 2, wherein the inlet side
channel, outlet side channel, and separation-use narrow channel are built
into the chip, an inlet connecting to the inlet side channel is present
on the chip surface, an outlet connecting to the outlet side channel is
present, and an opening connecting to an air chamber is present.

18. The micro channel chip according to claim 2, wherein the inner wall
of each channel is surface treated with a coating to prevent cell
adhesion or with a coating to prevent nonspecific adhesion.

19. The method according to claim 10, wherein the sample containing the
nonnucleated red blood cells and nucleated red blood cells is a fraction
that has been recovered with a density of 1.070 g/mL to 1.095 g/mL by
density gradient centrifugation separation using Percoll.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to Japanese Patent
Application No. 2009-205343 filed on Sep. 4, 2009, which is expressly
incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to a chip with a micro channel for
concentrating a granular material, to a chip for concentrating and
recovering nucleated red blood cells, and to a method for concentrating
and recovering nucleated red blood cells.

BACKGROUND ART

[0003] Conventional prenatal genetic testing methods not only place
physical and mental burdens on the mother, but also entail unavoidable
risks of injury and miscarriage of the fetus. Fetal cells (nucleated red
blood cells derived from the fetus) are known to travel in blood
circulating through the mothers body. If the fetal nucleated red blood
cells that are contained in maternal blood could be selectively recovered
and the fetal genes analyzed, it would be possible to conduct safe
prenatal diagnosis free of risk of injury and miscarriage to the fetus.
Using such a method, it would be possible to diagnose fetal genes in the
early period of pregnancy, and prepare early on for treatment. Worldwide,
about five million prenatal genetic diagnoses are performed each year.
Were it possible to practically develop such a safe genetic testing
method, it could be anticipated to occupy a large portion of the world
market.

[0004] However, fetal nucleated red blood cells, which are said to be
present in a quantity of about one cell per mL of maternal blood, are not
readily recovered. Methods of recovery, such as the use of an antibody
recognizing the unique structure of the surface of nucleated red blood
cells (an antigen-antibody reaction) and the use of
fluorescence-activated cell sorting (FAGS) employing fluorescence-labeled
blood cells, are being implemented by the research facilities of various
nations, but have all proven inadequate.

[0005] One method of recovering nucleated red blood cells that would
conceivably afford high reliability is to analyze an image under an
optical microscope and recover the nucleated red blood cells detected.
However, there is a problem in that a great amount of time is required to
detect the single nucleated red blood cell among the several billion
cells present in a single mL of blood.

[0006] It is anticipated that this problem can be solved by preprocessing
a blood sample that has been collected to separate and concentrate the
nucleated red blood cells. Currently, inventions and research in which
micro-sized structures (pillar structures, pore structures, and the like)
are fabricated and used to separate and concentrate the targeted cells
(Patent References 1, 2, 3; Non patent References 1, 2, 3) have been
reported as methods of separating blood cells using a physical structure
as a filter.

PRIOR ART REFERENCES

Patent References

[0007] [Patent Reference 1] Published Japanese Translation (TOKUHYO) No.
2009-509143 of a PCT International Application (WO2007/035585) [0008]
[Patent Reference 2] Published Japanese Translation (TOKUHYO) No,
2008-538283 of a PCT International Application (WO2006/108101) [0009]
[Patent Reference 3] Published Japanese Translation (TOKUHYO) No.
2006-501449 of a PCT International Application (WO2004/029221)

[0013] Patent References 1 to 3 and Nonpatent References 1 to 3 are hereby
incorporated in their entirety by reference.

SUMMARY OF THE INVENTION

Problem to be Solved by the Invention

[0014] In prior art such as that cited above, although the targeted cells
are separated or concentrated within the chip, no mechanism for
effectively recovering the targeted cells is present within the chip.

[0015] For example, in the chip described in Nonpatent Reference 3, cells
are separated based on size and cell deformation. There are channels
narrowing into four sections 15, 10, 5 and 2.5 μm in width (with a
constant depth of 5 μm). When a blood sample containing nucleated red
blood cells that are about 8 to 13 μm in diameter is made to flow
through the chip, the nucleated red blood cells pass through the channels
of width 15 μm, 10 μm, and 5 μm. However, they cannot pass
through the 2.5 μm channel, and are held in the first row of 2.5 μm
channels. In tests conducted with cord blood, which contains a higher
density of nucleated red blood cells than maternal blood, when the first
row became clogged with nucleated red blood cells, subsequently arriving
nucleated red blood cells passed through an evacuation passage provided
between two channels of 2.5 μm, and were recovered by a recovery
vessel provided at an exit. This procedure requires 6 to 8 hours. There
is no technique for recovering the nucleated red blood cells that have
been retained in the first row of 2.5 μm channels, so the recovery
efficiency of nucleated red blood cells is extremely poor. Maternal blood
contains quite few fetal nucleated red blood cells (on average, about 1.2
cells per mL). When this chip is employed, it is necessary to recover the
nucleated red blood cells that have lodged in the first row, but no
recovery method is indicated.

[0016] Accordingly, one object of the present invention is to provide a
chip having a structure that selectively concentrates fetal nucleated red
blood cells contained in maternal blood, and permits the recovery of a
concentrated liquid rich in nucleated red blood cells; and to provide a
chip permitting the recovery of specific granular material from a mixture
of granular materials of similar dimensions and differing deformation
properties. Another object of the present invention is to provide a
method for concentrating and recovering nucleated red blood cells by
concentrating nucleated red blood cells from maternal blood and
recovering a concentrated liquid rich in nucleated red blood cells.

Means of Solving the Problem

[0017] [1]

[0018] A micro channel chip, employed to concentrate a granular material B
from a mixture of at least one type of granular material having arbitrary
grain diameter and arbitrary deformation property (referred to
hereinafter as granular material A) and at least one type of granular
material having a larger grain diameter than granular material A and less
deformability than granular material A (referred to as granular material
B hereinafter),

[0019] comprising an inlet side channel, an outlet side channel, and a
separation-use narrow channel between the inlet side channel and outlet
side channel;

[0020] wherein the separation-use narrow channel has an inner wall of
dimensions permitting the ready passage of granular material A and
tending not to pass granular material B; and

[0021] comprising a means of deforming or displacing a part of the inner
wall of the channel to achieve dimensions facilitating the passage of
granular material B.

[0023] comprising an inlet side channel, an outlet side channel, and a
separation-use narrow channel between the inlet side channel and outlet
side channel;

[0024] wherein the separation-use narrow channel has an inner wall of
dimensions permitting the ready passage of nonnucleated red blood cells
and tending not to pass nucleated red blood cells; and

[0025] comprising a means of deforming or displacing a part of the inner
wall of the channel to achieve dimensions facilitating the passage of
nucleated red blood cells.

[3]

[0026] The micro channel chip according to [2], wherein the inner wall of
the separation-use narrow channel has a vertical sectional height in the
channel in a range of 1 μm to 5 μm, a width in a range of 5 μm
to 10 m, and a channel length in a range of 2 μm to 1 m.

[0028] comprising an inlet side channel, an outlet side channel, and a
separation-use narrow channel between the inlet side channel and outlet
side channel;

[0029] wherein the separation-use narrow channel has an inner wall of
dimensions permitting the ready passage of nonnucleated red blood cells
and tending not to pass nucleated red blood cells; and

[0030] wherein the dimensions are such that the vertical sectional height
in the channel is in a range of 1 μm to 5 μm, the width is in a
range of 5 μm to 10 m, and the length of the channel is in a range of
2 μm to 1 m.

[5]

[0031] The micro channel chip according to any one of [1] to [3], wherein
a plurality of separation-use narrow channels are separated by spacers,
the surface of the spacer facing the channel on the outlet side is a
curved surface that is convex in shape on the outlet side channel side,
and/or the surface of the spacer facing the channel on the inlet side is
a curved surface that is convex in shape on the inlet side channel side.

[6]

[0032] The micro channel chip according to any one of [1] to [3], [5],
wherein the means of deforming or displacing the inner wall of the
separation-use narrow channel is comprised of a flexible film disposed as
at least part of the inner wall of the separation-use narrow channel, and
a pressure-adjustable chamber disposed on the opposite side of the
channel from the flexible film.

[7]

[0033] The micro channel chip according to [6], wherein the inlet side
channel, outlet side channel, and separation-use narrow channel are built
into the chip, an inlet connecting to the inlet side channel is present
on the chip surface, an outlet connecting to the outlet side channel is
present, and an opening connecting to an air chamber is present.

[8]

[0034] The micro channel chip according to any one of [1] to [7], wherein
the inner wall of each channel is surface treated with a coating to
prevent cell adhesion or with a coating to prevent nonspecific adhesion.

[9]

[0035] A method for recovering a liquid in which nucleated red blood cells
have been concentrated, comprising:

[0036] feeding a sample containing nonnucleated red blood cells and
nucleated red blood cells from the inlet side channel of the micro
channel chip according to any one of [2] to [3], and [5] to [8];

[0037] recovering the liquid that has passed through the separation-use
narrow channel from the outlet side channel;

[0038] causing a part of the inner wall of the channel to deform or
displace to assume dimensions allowing ready passage of the nucleated red
blood cells,

[0039] and while in this state, feeding a recovering liquid from the inlet
side channel to recover a liquid rich in nucleated red blood cells from
the outlet side channel.

[10]

[0040] A method for recovering a liquid in which nucleated red blood cells
have been concentrated, comprising

[0041] feeding a sample containing nonnucleated red blood cells and
nucleated red blood cells to the inlet side channel of the micro channel
chip according to [4];

[0042] recovering the liquid that has passed through the separation-use
narrow channel from the outlet side channel;

[0043] feeding a recovering liquid from the inlet side channel or outlet
side channel, and

[0044] recovering a liquid rich in nucleated red blood cells from the
outlet side channel or inlet side channel.

[11]

[0045] The method according to [9] or [10], wherein the sample containing
the nonnucleated red blood cells and nucleated red blood cells is a
fraction that has been recovered with a density of 1.070 to 1.095 g/mL by
density gradient centrifugation separation using Percoll.

[12]

[0046] The method according to [11], wherein the sample containing the
nonnucleated red blood cells and nucleated red blood cells comprises a
recovered fraction diluted with a saline solution having a sodium
chloride concentration consistent with physiological conditions.

[13]

[0047] The method according to any one of [9], [11] to [12], wherein the
micro channel chip according to [6] or [7] is employed, and, in the
course of feeding the sample containing nonnucleated red blood cells and
nucleated red blood cells, the pressure-adjustable chamber is subjected
to a positive pressure relative to the separation-use narrow channel to
prevent a part of the inner wall of the channel from deforming in a
concave manner to the air chamber side.

[14]

[0048] The method according to any one of [9], [11] to [13], wherein the
micro channel chip according to [6] or [7] is employed and the pressure
in the air chamber is reduced relative to the separation-use narrow
channel to cause a part of the inner wall of the channel to deform or
displace in a concave manner to the air chamber side to achieve
dimensions permitting the ready passage of nucleated red blood cells.

[15]

[0049] The method according to any one of [9], [11] to [14], wherein the
micro channel chip according to [6] or [7] is employed and the pressure
in the air chamber is reduced relative to the separation-use narrow
channel to cause a part of the inner wall of the channel to deform or
displace in a concave manner to the air chamber side to achieve
dimensions permitting the ready passage of the liquid through the
separation-use channel when the liquid is introduced, during cleaning, or
when removing air bubbles.

Effects of the Invention

[0050] The present invention permits the concentration and recovery with
high efficiency of the nucleated red blood cells present at extremely low
concentration in maternal blood.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 Shows an exploded view descriptive of the three-layer
structure of one aspect of the chip of the present invention and an
enlarged view of channel-forming layer A.

[0052] FIG. 2 Shows a sectional view of a surface parallel to the channel
descriptive of the three-layer structure of one aspect of the chip of the
present invention.

[0053] FIG. 3 Schematic descriptive views of working of the intermediate
film and the separation of blood cells. The figure on the left shows the
air chamber in a slightly positively pressurized state, and the figure on
the right shows the air chamber in a negatively pressurized state.

[0054] FIG. 4 An image of the gap portion of a channel casting mold made
of SU-8 as observed by scanning electron microscopy in Embodiment 1. This
photograph is the casting mold; the actual channels are the reverse of
the protrusions and indentations.

[0056] FIG. 6 Photographs showing density gradient centrifugal separation
in Embodiment 2. The left photograph shows the introduction of maternal
blood diluted two-fold with physiological saline after layering 1.075
g/mL and 1.085 g/mL Percoll solutions in a test tube. The center
photograph was taken after centrifugal separation, with fractionation
into various specific gravities. The right photograph shows the recovery
of fractions corresponding to specific gravities primarily containing
nucleated red blood cells and neutrophils, diluted two-fold with
physiological saline.

[0057] FIG. 7 Photographs showing density gradient centrifugal separation
in Embodiment 2. The left photograph shows the right photograph in FIG. 6
following centrifugal separation. The right photograph was taken after
removing the layer containing a large amount of Percoll.

[0059] FIG. 9 A descriptive drawing showing working of the intermediate
film and the release of nucleated red blood cells retained in gaps in the
concentration and recovery of nucleated red blood cells in Embodiment 2.

[0060] FIG. 10 The appearance (left) of a PDMS chip employed in the
concentration and recovery of nucleated red blood cells in Embodiment 2.
The right figure is an image of blood cells retained in gaps as observed
by microscope after feeding the blood cell sample solution.

[0061] FIG. 11 Images showing the concentration and recovery of nucleated
red blood cells in Embodiment 2. The left figure is the image of blood
cells that have passed through the gaps as observed by microscope after
feeding the blood cell sample solution. The right figure is an image as
observed by microscope of how blood cells that have remained in the gap
are released by causing the intermediate film to work.

[0063] The present invention is a micro channel chip, employed to
concentrate a granular material B from a mixture of at least one type of
granular material having arbitrary grain diameter and arbitrary
deformation property (referred to hereinafter as granular material A) and
at least one type of granular material having a larger grain diameter
than granular material A and less deformability than granular material A
(referred to as granular material B hereinafter). This micro channel chip
comprises an inlet side channel, an outlet side channel, and a
separation-use narrow channel between the inlet side channel and outlet
side channel. The separation-use narrow channel has an inner wall of
dimensions permitting the ready passage of granular material A and
tending not to pass granular material B. The micro channel chip also
comprises a means of deforming or displacing a part of the inner wall of
the channel to achieve dimensions facilitating the passage of granular
material B.

[0064] Using the micro channel chip, it is possible to concentrate and
separate granular material B from the mixture of granular materials A and
B set forth above. An example of the mixture of granular materials A and
B is blood. Specifically, an example of granular material A is
nonnucleated red blood cells and an example of granular material B is
nucleated red blood cells. Accordingly, one aspect of the micro channel
chip employed to concentrate granular materials of the present invention
is a micro channel chip for concentrating red blood cells that is used to
concentrate nucleated red blood cells from a mixture of nonnucleated red
blood cells and nucleated red blood cells.

[0065] In the micro channel chip for concentrating granular materials of
the present invention, the dimensions of the inner walls of the
separation-use narrow channel are such that granular material A passes
readily and granular material B tends not to pass. The dimensions of the
inner walls of the separation-use narrow channel can be determined as set
forth below, for example, so that granular material A passes readily and
granular material B tends not to pass.

[0066] For example, the dimensions of the inner walls can be denoted as
follows. Let h denote the vertical sectional height in the channel. Let w
denote the width and let L denote the length of the channel. Denoting the
particle diameter of granular material A as φA and the deformation
property thereof as dfA, and denoting the particle diameter of granular
material B as φB and the deformation property thereof as dfB, the
height h, an important element in the performance of the chip, can be set
by satisfying the following conditions.

Height h>φA×dfA×k1

Height h<φB×dfB×k1

Here; k1 is a coefficient such that when the case where dfA and dfB
remain invariable is denoted as 1, it is possible to set k=1. Particle
diameters φA and φB can be suitably selected by known methods,
dfA and dfB are primarily determined by the flow rate of the sample
liquid in the chip and by the dimensions of the separation-use narrow
channel inlet, and can be determined taking into account the stress
applied to the granular materials in the separation-use narrow channel
inlet. Specifically, dfA and dfB can be obtained by applying an actual
sample as a test product on a chip and observing under a microscope the
degree to which the granular material deforms in the separation-use
narrow channel inlet.

[0067] Further, the effect of width w on the ease of passage of granular
material A and the difficulty of passage of granular material B is not as
great as that of height h. To facilitate the passage of granular material
A, it suffices to satisfy at least the following condition:

Width w>φA×(1/dfA)×k2

In this formula, k2 is at least 1. For example, it is desirably 2 to 30.
k2 can also be a value greater than 30.

[0068] For example, in the micro channel chip for concentrating nucleated
red blood cells, granular material A corresponds to nonnucleated red
blood cells and granular material B corresponds to nucleated red blood
cells. The particle diameter of nonnucleated red blood cells (granular
material A) is 4 to 6 μm. However, they are oblate particles about 2
μm in thickness. A thickness of about 2 μm is adopted as φA,
and deformation property dfA is 0.4 to 0.6. Nucleated red blood cells
(granular material B) vary in their particle diameter by stage. Those
handled by the present invention have a particle diameter B of about 8 to
13 μm and a deformation property dfB of about 0.7 to 0.9.

[0069] The present invention will be described below by means of the
example of a micro channel chip for concentrating nucleated red blood
cells.

[The Micro Channel Chip for Concentrating Nucleated Red Blood Cells]

[0070] According to FIG. 1, the micro channel chip for concentrating
nucleated red blood cells of the present invention is described. The
right side of FIG. 1 is an exploded view descriptive of a chip 1 having a
three-layer structure comprised of a channel-forming layer A, an
intermediate film B, and an air chamber-forming layer C. Inverted upper
and lower surfaces views of channel-forming layer A are shown on the
upper left. An enlarged view in the vicinity of separation-use narrow
channel 30 of channel-forming layer A is shown on the lower left.

[0071] Chip 1 comprises an inlet side channel 10, an outlet side channel
20, and a separation-use narrow channel 30 between the inlet side channel
and the outlet side channel in channel-forming layer A. Separation-use
narrow channel 30 is a narrow channel with dimensions that facilitate the
passage of nonnucleated red blood cells and hinder the passage of
nucleated red blood cells. The diameter of nonnucleated red blood cells
is about 4 to 6 μm, while the diameter of nucleated red blood cells is
about 8 to 13 μm. Further, as is also described in Nonpatent Reference
3, cells such as erythrocytes are capable of deforming, and can thus pass
through passages that are narrower than the size given above.
Specifically, separation-use narrow channel 30 can have a vertical
sectional height in the narrow channel that falls within a range of 1
μm to 6 μm, a width falling within a range of 5 μm to 10 m, and
a length falling within a range of 2 μm to 1 m. The effect on the
separation of nucleated red blood cells of the height in particular among
the cross-sectional dimensions is great in the example shown in FIG. 1.
Based on the results of the experiments given in the embodiments, the
closer the height of the narrow channel is to 1 μm, the greater the
recovery rate of nucleated red blood cells becomes, and the closer it
approaches 5 μm, the greater the drop in the recovery rate of
nucleated red blood cells. The vertical sectional height in the narrow
channel desirably falls within a range of 1 to 2 μm. The width can
fall within a range of 10 μm to 10 cm, and the length of the channel
can fall within a range of 20 to 300 μm. In the micro channel chip for
concentrating nucleated red blood cells set forth above, it is possible
to cause not just nonnucleated red blood cells, but also white blood
cells, to pass through the separation-use narrow channel, and to separate
nucleated red blood cells from white blood cells.

[0072] As shown in FIG. 1, separation-use narrow channel 30 desirably
comprises a plurality of narrow channels 30a, 30b, 30c . . . 30j. For
example, separation-use channel 30 can be comprised of 5 to 20 narrow
channels. However, the number of narrow channels is not limited. By way
of example, the number of narrow channels can range from 1 to 20,000.

[0073] Plurality of separation-use narrow channels 30 are separated by
spacers 31. The surface 32a of spacer 31 that faces the outlet side of
channel 20 is a curved surface of convex shape on the outlet channel
side. The surface 32b of the spacer that faces the inlet side of channel
10 is a curved surface of convex shape on the inlet side of the channel.
This is desirable so that blood clots tend not to form on the inlet side
or outlet side of the spacer, and so as not to impede the passage of the
blood sample. Specifically, in terms of design, the curved surfaces of
convex shape are semicircles with diameters of 20 to 40 μm. Further,
the lateral surface 33b (the surface facing the inlet side of channel 10)
of the bank 33 for forming separation-use narrow channel 30 and the
lateral surface 33a (the surface facing the outlet side of channel 20,
not shown) can also be made similarly convex in shape (with lateral
surfaces 33b and 33a being of an overall undulating shape).

[0074] The inlet side of channel 10, the outlet side of channel 20, and
separation-use narrow channel 30 are build into chip 1. Inlet 11
connecting to the inlet side of the channel, outlet 21 connecting to the
outlet side of the channel, and opening 51 connecting to the air chamber
are present on the outer surface of chip 1. As shown in FIGS. 1 and 2
(sectional view), chip 1 can have a three-layer structure comprised of a
channel-forming layer A, an intermediate film B, and an air
chamber-forming layer C. On one surface, channel-forming layer A
comprises inlet side channel 10, outlet side channel 20, and
separation-use narrow channel 30 between the inlet side channel and
outlet side channel. On the other surface (opposing surface),
channel-forming layer A comprises an inlet 11 connecting to the inlet
side channel and an outlet 21 connecting to the outlet side channel. On
the other surface thereof, channel-forming layer A comprises an opening
51 connecting to the air chamber.

[0075] Intermediate film B can have the dimensions of a plane identical to
the planar dimensions of channel-forming layer A and air chamber-forming
layer C. An opening 41 can be present that connects the space between
opening 51 connecting to air chamber 50 present in channel-forming layer
A, and air chamber 50 present in air chamber-forming layer C.

[0076] Chip 1 also comprises a means of facilitating the passage of
nucleated red blood cells through separation-use narrow channel 30 by
deforming or displacing a part of the inner wall of narrow channels 30a,
30b, 30c . . . 30j of separation-use narrow channel 30. The means of
deforming or displacing the inner wall of the separation-use narrow
channel can be comprised of a flexible film 40 provided as at least a
part of the inner wall of the separation-use narrow channel and air
chamber 50 provided on the opposite side of the flexible film from the
channel. Flexible film 40, constituting a portion of intermediate film B,
functions as a diaphragm. Positive pressure is exerted on air chamber 50
relative to the channel side, causing flexible film 40 to press against
spacer 31 forming separation-use narrow channel 30 and controlling
separation-use narrow channel 30 to within the prescribed dimensions set
forth above. When the elasticity of intermediate film B itself and the
adhesion between the intermediate film and the spacer can maintain the
prescribed dimensions, it is possible not to positively pressurize air
chamber 50 relative to the channel side. In this state, nucleated red
blood cells either cannot pass through separation-use narrow channel 30,
or can do so only with difficulty. By contrast, when the pressure in air
chamber 50 is reduced, flexible film 40 warps to the air chamber 50 side,
the distance between flexible film 40 and the surface of separation-use
narrow channel 30 opposite flexible film 40 increases, and it becomes
easy for nucleated red blood cells to pass through separation-use narrow
channel 30.

[0077] The concentration and recovery of nucleated red blood cells using
the chip of the present invention will be further described using FIG. 3.
Chip 1 of the present invention comprises a narrow channel (micro gap) in
which is built a diaphragm drive mechanism comprised of an air chamber 50
and a flexible film 40 functioning as a diaphragm that is deformed by air
pressure controls on a portion of separation-use narrow channel 30. A
blood sample containing targeted cells (nucleated red blood cells) that
is collected from the mother passes through a micro channel having a
narrow channel (micro gap), such as that shown on the left in FIG. 3. The
nucleated red blood cells (being larger or tending not to deform as much)
have greater difficulty passing through the narrow channel than other
cells. Thus, the nucleated red blood cells can be selectively trapped at
the front of the narrow channel and separated (in case concentration is
incomplete, separated) from other cells (mainly nonnucleated red blood
cells) that pass more easily along the narrow channel. Subsequently, as
shown on the right in FIG. 3, flexible film 40 functioning as a diaphragm
is deformed by reducing the pressure in air chamber 50, making it
possible to recover the group of cells consisting of concentrated
nucleated red blood cells containing the nucleated red blood cells that
have been trapped in the front of the narrow channel.

[0078] It is suitable for flexible film 40 to be able to keep the narrow
channel to within prescribed dimensions in the course of selectively
trapping nucleated red blood cells in the front of the narrow channel by
increasing the pressure relative of air chamber 50 relative to the
channel side, and for flexible film 40 to have physical properties that
impart to the narrow channel a gap adequate to allow nucleated red blood
cells to pass through the narrow channel when the pressure has been
reduced in air chamber 50. From such perspectives, flexible film 40 (or
intermediate film B) can be suitably made of silicone resin to impart
suitable elasticity and firmness, for example. The phrase "suitable
elasticity and firmness" means, for example, resistance to deformation
adequate to maintain the dimensions of the gap in the course of
generating positive pressure relative to the channel side, and adequate
deformation to permit recovery of the nucleated red blood cells in the
course of generating negative pressure relative to the channel side.
Accordingly, such adequate elasticity and firmness are values that depend
on the gap between spacer 31 and spacer 31, and on the size and shape of
air chamber 50. The elasticity and firmness of a silicone resin film also
varies with the thickness of the film. Thus, it is possible to obtain a
film of the desired elasticity and firmness by adjusting the film
thickness of the silicone resin in a single material. An example of a
silicone resin is polydimethyisiloxane. The gap between spacers 31 is 30
μm, and when air chamber 50 is adequately large, the film thickness
can be kept to within a range of 20 to 200 μm, for example.

[0079] The inner walls of each channel can be surface treated with a
coating agent to prevent cell adhesion or a coating agent to prevent
non-specific adhesion, for example. This surface treatment inhibits the
adhesion and aggregation of blood cells, platelets, proteins, and the
like on the inner walls of the various channels and facilitates the above
separation. Examples of coating agents for preventing non-specific
adhesion are Blockmaster CE-510 (JSR K.K.), comprising a chief component
in the form of polyethylene glycol (PEG), and Lipidure (NOF Corp.).

[0080] The present invention covers micro channel chips for concentrating
nucleated red blood cells comprising an inlet side channel, an outlet
side channel, and a separation-use narrow channel between the inlet side
channel and outlet side channel, in which the inner walls of the
separation-use narrow channel are of dimensions that facilitate the
passage of nonnucleated red blood cells and hinder the passage of
nucleated red blood cells, and in which these dimensions are such that
the vertical sectional height in the channel falls within a range of 1
μm to 5 μm, the width falls within a range of 5 μm to 10 m, and
the length of the channel falls within a range of 2 μm to 1 m. The
inlet side channel, outlet side channel, and separation-use narrow
channel in micro channel chips of this aspect are identical to the micro
channel chip for concentrating nucleated red blood cells of the aspect
set forth above. The fact that the dimensions of the inner walls of the
separation-use narrow channel facilitate the passage of nonnucleated red
blood cells and hinder the passage of nucleated red blood cells, and the
fact that these dimensions are such that the vertical sectional height in
the channel falls within a range of 1 μm to 5 μm, the width falls
within a range of 5 μm to 10 m, and the length of the channel falls
within a range of 2 μm to 1 m are identical for the micro channel chip
for concentrating nucleated red blood cells of the aspect set forth
above.

[0081] However, the chip of this aspect does not have a means based on a
method of deforming or displacing a part of the inner wall of the channel
to facilitate the passage of nucleated red blood cells. When employing a
chip of this aspect, the concentration and recovery of nucleated red
blood cells are accomplished by feeding a sample containing nonnucleated
red blood cells and nucleated red blood cells through an inlet side
channel of the micro channel chip, recovering the liquid that passes
through the separation-use narrow channel from the outlet side channel,
and then feeding the recovering liquid from the inlet side channel or
outlet side channel to recover a liquid rich in nucleated red blood cells
from the outlet side channel or inlet side channel. That is, the chip of
this aspect permits the recovery of a liquid of concentrated nucleated
red blood cells by the above method despite not having a means based on a
method of facilitating the passage of nucleated red blood cells by
deforming or displacing a part of the inner walls of the channel.

[0082] Various techniques can be employed to manufacture the chip of the
present invention. The technique that is employed is selected in part
based on optimal materials. Typical materials for manufacturing the chip
of the present invention are glass, silicon, steel, nickel,
polymethylmethacrylate (PMMA), polycarbonate, polystyrene, polyethylene,
polyolefin, silicons (for example, polydimethylsiloxane), and
combinations thereof. Additional materials are known in the technical
field. Methods of manufacturing channels from these materials are known
in the technical field. These methods include photolithography (such as
3D lithography and X-ray photolithography); molding methods; embossing
methods; silicon micromachining methods; wet and dry chemical etching
methods: milling methods; diamond cutting methods; lithography,
electroplating, and molding (LIGA) methods; and electroplating. For
example, with glass, traditional photolithography silicon manufacturing
methods followed by wet (KOH) or dry etching (reactive ion etching
employing fluorine or some other reactive gas) can be employed.
Techniques such as laser microprocessing methods can be adopted for
plastic materials with high light absorption efficiency. Since the
process in this technique is a continuous one, it is suited to low
throughput manufacturing. For mass-produced plastic chips, the
thermoplastic injection molding method and compression molding method are
suitable. To manufacture the chip of the present invention, the
conventional thermoplastic injection molding that is employed to mass
produce compact disks (while retaining functional fidelity at the
sub-micron level) can be utilized. For example, the functions of the chip
can be replicated on a glass master by conventional photolithography.
When the glass master is electrically cast, a sturdy, thermal
shock-resistant, thermoconductive, hard mold can be produced. This mold
then serves as a master template in injection molding or compression
molding to mold these functions into plastic chips. Compression molding
or injection molding can be selected as the manufacturing method based on
the plastic material to be used to manufacture the chip, as well as based
on conditions relating to the optical quality and throughput of the final
product. The compression molding method (also referred to as the hot
embossing method or relief imprinting method) is excellent for small
structures, but is difficult to use and has a long cycle time when
replicating structures with high vertical/horizontal ratios. However, it
affords the advantage of being suited to high molecular weight polymers.
The injection molding method works well even on structures with high
vertical/horizontal ratios, and is optimal for low molecular weight
polymers.

[0083] The chip can be manufactured as a single unit or as multiple pieces
that are subsequently assembled. In one aspect, each of the layers of
channel-forming layer A, intermediate film B, and air chamber-forming
layer C of the chip has a channel, through-hole, or the like, as shown in
FIG. 1. The layers of the chip can be bonded together by clamps,
adhesives, heat, anodic bonding, or surface bonding (such as wafer
bonding). Further, chips having channels on more than one flat surface
can be fabricated as single pieces by 3D lithography or some other 3D
manufacturing technique.

[0085] The present invention includes a method for preparing a
concentrated solution of nucleated red blood cells. This method for
preparing a concentrated solution of nucleated red blood cells employs
the micro channel chip of the present invention set forth above.
Specifically, it is a method comprising:

(1) feeding a sample containing nonnucleated red blood cells and
nucleated red blood cells from the inlet side channel of the chip and
recovering the liquid that has passed through the separation-use narrow
channel from the outlet side channel; and then (2) causing a part of the
inner wall of the channel to deform or displace to assume dimensions
allowing ready passage of the nucleated red blood cells, and while in
this state, feeding the recovering liquid from the inlet side channel to
recover a liquid rich in nucleated red blood cells from the outlet side
channel.

[0086] The sample containing nonnucleated red blood cells and nucleated
red blood cells is a blood sample containing the targeted cells
(nucleated red blood cells) that has been collected from the mother. The
size of the blood sample that is collected each time is normally about 5
to 10 mL. In the present invention, the entire quantity or some portion
thereof can be employed to recover a liquid rich in nucleated red blood
cells.

[0087] From the perspective of efficiently recovering nucleated red blood
cells, the sample containing nonnucleated red blood cells and nucleated
red blood cells is desirably fractionated to obtain a fraction with a
high concentration of nucleated red blood cells prior to concentration
processing with the chip. The fraction with a high concentration of
nucleated red blood cells can be a fraction that has been recovered with
a density of 1.070 g/mL to 1.095 g/mL, or 1.075 g/mL to 1.885 g/mL by,
for example, density gradient centrifugal separation using Percoll, for
example. For the former range, nucleated red blood cells can be recovered
from a broad range, while for the latter range, which is narrower than
the former range, the ratio of nonnucleated red blood cells present with
nucleated red blood cells decreases, enhancing separation efficiency.
However, there is no intention to limit the present invention thereto. In
addition, methods such as the Ficoll method and red blood cell
agglutination method can also be employed to obtain a fraction with a
high concentration of nucleated red blood cells. Cells that are larger
than or less prone to deformation than nucleated red blood cells, such as
white blood cells, are desirably removed in advance by a suitable method.
To that end, the dimensions employed in the present invention can be
varied.

[0088] From the perspective of inhibiting clogging of the narrow channels
of the chip by blood cells and inhibiting adhesion of blood cells on the
inner walls of the channels, the sample containing nonnucleated red blood
cells and nucleated red blood cells is suitably obtained by diluting a
fraction that has been recovered with a saline solution having a sodium
chloride concentration consistent with physiological conditions. The
sodium chloride concentration consistent with physiological conditions
falls within a range of 8 to 10 mg/mL, for example. The degree of
dilution with a saline solution can be suitably determined by taking into
account the flow rate, channel structure, and the like. It is suitable to
dilute to a blood cell concentration falling within a range of
1.1×106 to 2.3×106 cells/μL.

[0089] The operation of feeding a sample containing nonnucleated red blood
cells and nucleated red blood cells from the inlet side channel of the
chip and recovering the liquid that has passed through the separation-use
narrow channel from the outlet side channel can be conducted by setting
the sample feed rate (flow rate) to, for example, within a range of 1 to
100 μL/minute. The sample can be fed using a micro syringe pump
(IC3100/KN3319040 Tech-jam) or the like.

[0090] The micro channel chip is suitably the chip of the present
invention. Specifically, it is suitable to employ a chip in which the
means of causing the inner wall of the separation-use narrow channel to
deform or displace is comprised of a flexible film provided on at least a
part of the inner wall of the separation-use narrow channel and an air
chamber provided on the opposite side of the flexible film from the
channel; an inlet side channel, outlet side channel, and separation-use
narrow channel are built into the chip; and having on the surface of the
chip, an inlet connecting to an inlet side channel, an outlet connecting
to an outlet side channel, and an opening connecting to the air chamber.
Thus, in the course of feeding a sample containing nonnucleated red blood
cells and nucleated red blood cells, if necessary, positive pressure
relative to the channel side is applied to the air chamber, preventing
part of the inner wall of the channel from deforming or displacing in a
concave manner to the air chamber side.

[0091] Once recovery of the liquid that has passed through the
separation-use narrow channel has been completed, a part of the inner
wall of the channel is caused to deform or displace to assume dimensions
allowing ready passage of the nucleated red blood cells, and while in
this state, the recovering liquid is fed from the inlet side channel to
recover a liquid rich in nucleated red blood cells from the outlet side
channel. Specifically, in the micro channel chip, the pressure in the air
chamber is reduced to cause a part of the inner wall of the channel to
deform or displace in a concave manner to the air chamber side and assume
dimensions facilitating the passage of the nucleated red blood cells. A
liquid rich in nucleated red blood cells can be recovered, for example,
by employing a saline solution having a sodium chloride concentration
consistent with physiological conditions that is employed in the above
dilution.

[0092] The liquid rich in nucleated red blood cells that is recovered can
be used in fetal diagnosis and the like. More specifically, the blood
cells are stained by the May-Grunwald-Giemsa staining protocol. To a
preparation used for microscopic observation, solution is added dropwise
in 2.5 μL quantities. It is then smeared with a glass slide and dried.
Subsequently, it is stained by immersion in a glass vessel filled with
the stain solution, washed, and dried. The nucleated red blood cells are
selected morphologically by observation under a microscope and recovered.
Finally, the DNA of the nucleated red blood cells is extracted and
genetically analyzed.

[0093] The micro channel chip for concentrating granular material of the
present invention is not limited to the concentration of nucleated red
blood cells. It can be similarly employed to separate and concentrate
granular materials of differing size, firmness, and ability to deform. In
particular, it can be used to separate and recover leukocytes and
erythrocytes. In that case, the red blood cells correspond to granular
material A (at least one type of granular material of arbitrary particle
diameter and arbitrary deforming property) and the white blood cells
correspond to granular material B (at least one type of granular material
having a particle diameter greater than that of granular material A and
an ability to deform less than that of granular material A). Multiple
chips of the present invention with gaps of different dimensions can be
used to recover various fractions by connecting them in series. It is
particularly effective to separately provide a micro channel chip for
concentrating granular material having a gap that does not pass cells
that are larger than nucleated red blood cells, particularly leukocytes,
upstream from the chip of the present invention. However, as set forth
above, the micro channel chip of the present invention can be used to
cause the separation-use narrow channel to pass not just nonnucleated red
blood cells, but also white blood cells (at which time, nucleated red
blood cells do not pass through the separation-use narrow channel). As a
result, it is also possible to separate nucleated red blood cells from
white blood cells (see Embodiment 3).

[0094] In the course of concentration and recovery using the chip of the
present invention, it is sometimes necessary to initially introduce a
liquid because the micro channels will normally be filled only with air.
In this process, air bubbles often form in narrow portions, In that case,
they remain in the vicinity of separation-use narrow channel 30, the
liquid takes a long time to surmount the narrow portions, and powerful
pressure is sometimes needed. In the present invention, at such times, it
is possible to deform the diaphragm and readily introduce the liquid.

[0095] As set forth above, the present invention includes chips without a
means based on a method of deforming or displacing a part of the inner
wall of the channel to facilitate the passage of nucleated red blood
cells. When employing a chip of that form, nucleated red blood cells can
be concentrated and recovered by feeding a sample containing nonnucleated
and nucleated red blood cells from the inlet side channel of the micro
channel chip, recovering the liquid that has passed through the
separation-use narrow channel from the outlet side channel, feeding the
recovering liquid from the inlet side channel or outlet side channel, and
recovering a liquid rich in nucleated red blood cells from the outlet
side channel or inlet side channel. The recovering liquid and the sample
containing nonnucleated red blood cells and nucleated red blood cells are
identical to those set forth above.

EMBODIMENTS

[0096] The present invention is described in greater detail below through
embodiments. However, there is no intent to limit the present invention
to the embodiments.

EMBODIMENT 1

Chip Preparation

[0097] The series of steps up to completion of a chip of
polydimethylsiloxane (PDMS) can be roughly divided into designing the
channel pattern, preparation of a mask for photolithography, preparation
of a channel casting mold, preparation of a PDMS channel layer employing
the casting mold, and bonding the various layers.

Designing the Channel Pattern

[0098] The channel pattern was constructed with Illustrator (Adobe) on a
PC.

Preparation of a Mask for Photolithography

[0099] The channel pattern was printed on a transparent OHP film. This
film was adhered to a transparent glass sheet and a mask for
photolithography was completed.

Preparation of a Channel Casting Mold

[0100] Photocuring resist SU-8 (US Micro Chem Corp.) was employed as the
material of the channel casting mold. A six-inch silicon wafer with a
single surface polished to a mirror finish (CZ-N, Shin-Etsu Chemical Co.,
Ltd., 625 μm in thickness, crystal plane <100>) was employed as
the substrate. The surface of the substrate was cleaned with ultrapure
water, dried with nitrogen gas, washed with acetone (EL grade, Kanto
Chemical Co., Inc.), and dried again with nitrogen gas. Following
cleaning, the substrate was secured to a spin coater (1H-DX2, Mikasa
K.K.), a suitable quantity of SU-8 was applied dropwise to the
mirror-finished surface side, and spin coating was conducted. Next, the
substrate that had been coated with SU-8 was placed on a hot plate
(DATAPLATE, AS ONE Corp.), heated for 1 minute at 65° C., and then
heated for 1 minute at 95° C. The power to the hot plate was then
cut, and the substrate was left standing until the substrate temperature
had dropped to about room temperature (pre-baking). A glass mask having
the above-described channel pattern was set on a contact exposure-type
mask aligner (Suss MJB3 UV400, made by Karl Suss Corp. of the US). UV
radiation with a wavelength of 365 nm (i line) was then irradiated onto
the SU-8 coated side of the substrate. Following UV exposure, the
substrate was placed on the hot plate, heated for 1 minute at 65°
C., and heated for 1 minute at 95° C., at which time the power to
the hot plate was cut. The substrate was left standing until the
substrate temperature had dropped to about room temperature
(post-baking). Next, the substrate was immersed for 1 minute 30 seconds
in developer (SU-8 Developer, MICRO CHEM) to remove portions of the SU-8
that had not been exposed to UV through the mask. The substrate was then
dried with nitrogen gas to complete a channel casting mold of SU-8.

Preparation of a PDMS Channel Layer Employing the Casting Mold

[0101] A SYLGARD 184 silicone elastomer kit (Dow Corning Toray Corp.) was
employed with polydimethylsiloxane (PDMS), a silicone resin, as the
material. The PDMS main material and a crosslinking agent were mixed in a
weight ratio of 10:1. To remove the air that had been entrained during
mixing, the PDMS was placed in a bell jar and a vacuum was drawn with a
rotary vacuum pump until the bubbles had been completely removed. The
debubbled PDMS was caused to flow into an SU-8 channel casting mold on
which had been mounted a frame made of silicone rubber to prevent the
PDMS from flowing out into the surroundings. Next, it was placed in an
oven (DKN 301, Yamato Scientific Co., Ltd.) and left standing for 5 hours
at 65° C. to cure the PDMS. The cured PDMS channel was carefully
separated from the channel casting mold. A belt hole puncher that had
been cleaned with acetone was used to punch holes in necessary spots in
the PDMS channel to serve as mounting holes for tubes for controlling the
air pressure and for a reservoir for introducing solution.

Bonding the Various Layers

[0102] To remove impurities such as dust that adhered to the PDMS, it was
washed with ultrapure water and dried with nitrogen gas. A B layer and a
C layer, the surface of which had been cleaned, were oxygen plasma
treated for 10 s at 8.8 Pa, 100 W, and 100 sccm with a reactive ion
etching apparatus (Reactive Ion Etching-10NR, SAMCO Inc.). While being
careful not to touch the surface that had been rendered hydrophilic by
the plasma treatment, tweezers were used to handle layers B and C, and a
PDMS chip onto which they had been bonded was completed.

[0103] As shown in FIGS. 1 and 2, a chip with a three-layer structure
comprised of a channel layer, a thin-film layer (intermediate film
layer), and an air layer was prepared. In FIG. 1, air inlet 51 of air
chamber 50 is provided on the same surface as inlet 11 of channel 10 and
outlet 21 of channel 20. By contrast, in FIG. 2, the air inlet 51 of air
chamber 50 is provided on the opposite surface from the inlet 11 of
channel 10 and the outlet 21 of channel 20. FIG. 2 is a sectional view of
the surface parallel with the channel to describe the three-layer
structure of the chip. A channel for introducing maternal blood was
microprocessed in layer A. Layer B is deformed by applying positive
pressure or negative pressure relative to the channel side to the air
chamber portion of layer C, and moves vertically in the gap portion in
the center of the figure.

[0104] A minute gap (microgap) 30 was fashioned in the center of the
channel, Holes 5 mm in diameter were fashioned as inlet 11 of channel 10
and outlet 21 of channel 20, and a reservoir was prepared by bonding with
intermediate layer B.

[0105] FIG. 3 shows schematic descriptive views of working of the
intermediate film and the separation of blood cells. It also shows an
enlarged view of the vicinity of minute gap (microgap) 30. The figure on
the left in FIG. 3 shows the air chamber of layer C in a slightly
positively pressurized state relative to the channel, making it possible
to prevent the intermediate film of layer B from dropping by means of the
pressure generated by flowing of the solution. It shows how blood cells
other than nucleated red blood cells that tend to readily deform flow on
through the gap. The figure on the right in FIG. 3 shows the state of
release for recovering the nucleated red blood cells that have collected
by expanding the channel of the gap portion by dropping of the
intermediate film of layer B by reducing the pressure of the air chamber
of layer C.

[0106] As shown in FIG. 3, intermediate film B swings up and down based on
the air that is introduced into air chamber 50, and is part of the
diaphragm drive. The diaphragm drive is designed to be readily installed
by forming air inlet 51 of air chamber 50 on the lower side. The swinging
of intermediate film B by driving the diaphragm is achieved by means of
the air pressure introduced into air chamber 50.

[0107] FIG. 4 is an image of the gap portion of the channel casting mold
made of SU-8 as observed by scanning electron microscopy. This photograph
is of the casting mold. The actual channel is the reverse of these
indentations and protrusions. As shown in FIG. 4, a channel layer
branching into a total of 10 channels from a single channel has been
fabricated. The channel portions that branch into multiple channels were
prepared as gap portions with heights of 1.4 μm or less. The height of
the gaps must be such that the nucleated red blood cells that are to be
collected in the present embodiment retain. Accordingly, the height of
the gaps started at 2.5 μm or less. At a flow rate of 0.1 to 10
μL/min, the nucleated red blood cells retained when the gap height
became 1.4 μm. Based on the above, the height of the gaps in the chip
was made 1.4 μm or less. The channel spacers were fabricated to
inhibit the chip from flexing due to the minute gaps. The spacer inlets
in the gap portions were imparted with from square to round (round edged)
shapes to inhibit stagnation from occurring when the blood passed by.

EMBODIMENT 2

[0108] Nucleated red blood cells were concentrated and recovered from
maternal blood by the following method using the chip fabricated in
Embodiment 1.

<Method of Introducing Blood>

[0109] Time was required for the nucleated red blood cells to concentrate
on the chip with the quantities of blood collected. Thus, a step to
reduce the quantity of blood was necessary. Accordingly, density gradient
centrifugal separation was conducted to concentrate nucleated red blood
cells and reduce the quantity of blood

1. Density Gradient Centrifugal Separation

[0110] Maternal blood (6.0 mL to 7.0 mL) is collected with a pipette and
transferred in two parts to a centrifuge tube. The quantity of maternal
blood collected is affected by individual differences in blood viscosity
and varied somewhat. However, 6.0 mL or more is consistently collected.
The maternal blood that had been divided into two parts is diluted
two-fold with a 0.9% (g/mL) NaCl aqueous solution. In a separate
centrifuge tube, a density gradient is prepared with Percoll of differing
densities (1.075 g/mL, 1.085 g/mL). These results are given in FIG. 5 as
a photograph (left) of the test tube to which the material blood was
charged, a photograph (middle) taken when the maternal blood was diluted
two-fold with physiological saline, and a photograph (right) taken when
1.075 g/mL and 1.085 g/mL Percoll solutions were layered.

[0111] The maternal blood that had been diluted two-fold is poured into
the centrifuge tube prepared with a density gradient. Centrifugal
separation is conducted with a centrifuge (3,000 rpm, 1,750×g, 30
min). The nucleated red blood cell-containing layer that appeared
following centrifugation is recovered with a pipette. The nucleated red
blood cell-containing layer recovered by pipette is diluted two-fold with
a 0.9% (g/mL) NaCl aqueous solution. The results are given in FIG. 6 as a
photograph (left) taken when maternal blood is introduced that had been
diluted two-fold with physiological saline after layering 1.075 g/mL and
1.085 g/mL Percoll solutions in the test tube; a photograph (middle, with
fractionation into various specific gravities) taken following
centrifugal separation; and a photograph (right) taken when the fractions
corresponding to the specific gravities containing primarily nucleated
red blood cells and neutrophils were recovered and diluted two-fold with
physiological saline.

[0112] The nucleated red blood cell-containing layer that was recovered
after density gradient centrifugal separation and diluted two-fold with
0.9% (g/mL) NaCl aqueous solution contains a large quantity of Percoll.
Thus, most of the remaining Percoll is made to move into the top layer of
blood cells by conducting centrifugal separation again, and removed with
an aspirator. This Percoll removal method is called cleaning. Cleaning is
conducted three or more times. The results are given in FIG. 7 as a
photograph (left) taken after centrifugal separation of the sample in the
photograph on the right in FIG. 6, and a photograph (right) taken after
removing the Percoll-containing layer.

[0113] The total quantity of blood that had been cleaned and recovered was
reduced to about 30 to 60 μL. The entire quantity was reduced to a
small quantity. Since the number of blood cells per visual field had
decreased, the nucleated red blood cells can be said to have been
concentrated. The results are given in FIG. 8. The image on the left was
obtained by staining the whole blood by the May-Grunwald-Giemsa staining
protocol and observing the nucleated red blood cells under a microscope.
The image on the right was obtained by staining the blood cells following
Percoll centrifugal separation by the May-Grunwald-Giemsa staining
protocol and observing the nucleated red blood cells under a microscope,

[0114] Nucleated red blood cells are recovered with the chip fabricated in
Embodiment 1 by causing nucleated red blood cells to retain in the gap
portion of the channel in the form of the separation-use narrow channel,
causing nonnucleated red blood cells and the like to pass through (left,
FIG. 9), and subsequently working the intermediate film (right, FIG. 9).
The height of the gap portion was set to 1.0 μm by optimizing the
minute gap portion. The blood sometimes settled while passing through the
channel. A countermeasure to this was set forth in the optimization of
the microgap portion

2.2 Introducing the Blood onto the Chip

[0115] Maternal blood (30 to 60 μL) the quantity of which had been
decreased in 2.1 is diluted four-fold and introduced onto the chip. The
introduction rate is 10 μL/min. The blood that had passed to the
reservoir before working of the intermediate film was sequentially
recovered with a micro-pipette. FIG. 10 (left) shows the appearance of
the PDMS chip. The right shows an image obtained by feeding the blood
cell sample solution and observing the blood cells that retained in the
gap portion under a microscope.

[0116] To the blood cells that retained in the gap portion is added a 0.9%
(g/mL) NaCl aqueous solution and the intermediate film is worked to
recover the blood cells in the reservoir. FIG. 11 shows images at that
time. The image on the left was obtained by feeding the blood cell sample
solution and observing the blood cells that passed through the gap
portion under a microscope. The image on the right was obtained by
working the intermediate film to release the blood cells that had
retained in the gap and observing them under a microscope.

<The Blood Cells Following Introduction>

[0117] The blood cells that had retained in the gap portion inlet were
released and recovered from the reservoir. The recovered solution was
stained with Giemsa to prepare a specimen. FIG. 12 shows a photograph in
which nucleated red blood cells were confirmed by microscope. The arrows
indicate nucleated red blood cells.

[0118] The above operations were implemented with three chips with gap
(separation-use narrow channel) heights of 1.0 μm, 1.4 μm, and 1.85
μm, respectively. The number of nucleated red blood cells detected
decreased depending on the gap height. For gap (separation-use narrow
channel) heights of 1.0 μm, 1.4 μm, and 1.85 μm, the number of
retained nucleated red blood cells was 8 cells, 6 cells, and 3 cells,
respectively. The respective recovery rates were 8/8, 6/8, and 3/8. The
recovery rate of nucleated red blood cells exhibited its highest value
for a gap height of 1.0 μm. That is, the gap height that was
advantageous for retaining nucleated red blood cells was 1.0 μm or
less. The red blood cells in the samples recovered could not be
confirmed. That is thought to be because the red blood cells had passed
through the gap portions. For these reasons, it can be said that it was
possible to concentrate the nucleated red blood cells because far fewer
blood cells other than nucleated red blood cells were present following
introduction onto the chip than prior to introduction onto the chip.

EMBODIMENT 3

[0119] The white blood cell and red blood cell elimination rates were
determined using the chip of Embodiment 2. First, the original white
blood cell count and red blood cell count of 1 mL of maternal blood are
determined by FACS. Then, an identical 1 mL of maternal blood is passed
over a chip with a microgap of 1.0 μm and the white blood cell count
and red blood cell count of the solutions that passed through and are
recovered are determined by FACS. The number of blood cells in the
solution that passed through the gap is divided by the blood cell count
in the original maternal blood and multiplied by 100 to obtain the
passage rate (%). The capture rate is calculated as 100-the passing rate.
These are determined for the red blood cells and white blood cells. The
remaining conditions are identical to those in Embodiment 2.

[0120] The results are given below. The original red blood cell count in 1
mL of maternal blood was 3.63×109. The red blood cell count
following passage was 3.40×109. On this basis, the red blood
cell passage rate was 93.6% and the red blood cell capture rate was
6.34%. The white blood cell count in 1 mL of maternal blood was
1.66×107. The white blood cell count following passage was
1.64×107. On this basis, the white blood cell passage rate was
98.7% and the white blood cell capture rate was 127%.

[0121] It merits noting that white blood cells, which are much larger than
nucleated red blood cells, are eliminated at a quite high rate. That is
attributed to the fact that white blood cells have a live nucleus, are
richer in flexibility, and more readily deform than nucleated red blood
cells prior to enucleation.

[0122] Thus, the removal rate of leukocytes and erythrocytes was about 95%
in the present embodiment, making it possible to reduce the overall blood
cell count to about one in twenty. That indicates that it is possible to
shorten the time required for automated image processing to one-twentieth
the current level, which is a tremendous effect.

INDUSTRIAL APPLICABILITY

[0123] The present invention is useful in the fields of the manufacturing
and use of chips for concentrating nucleated red blood cells.